The cell cycle inhibitory protein p21cip is not essential for maintaining beta-cell cycle arrest or beta-cell function in vivo

p21(cip1), a regulatory molecule upstream of the G(1/0) checkpoint, is increased in beta-cells in response to mitogenic stimulation. Whereas p21(cip1) can variably stimulate or inhibit cell cycle progression, in vitro studies suggest that p21(cip1) acts as an inhibitor in the pancreatic beta-cell. To determine the functional role of p21(cip1) in vivo, we studied p21-null mice. Surprisingly, islet mass, beta-cell replication rates, and function were normal in p21-null mice. We next attempted to drive beta-cell replication in p21-null mice by crossing them with rat insulin II promoter-murine PL-1 (islet-targeted placental lactogen transgenic) mice. Even with this added replicative stimulus of PL, p21-null islets showed no additional stimulation. A G(1/S) proteome scan demonstrated that p21(cip1) loss was not associated with compensatory increases in other cell cycle inhibitors (pRb, p107, p130, p16, p19, and p27), although mild increases in p57 were apparent. Surprisingly, p18, which had been anticipated to increase, was markedly decreased. In summary, isolated p21(cip1) loss, as for pRb, p53, p18, and p27 and other inhibitors, results in normal beta-cell development and function, either because it is not essential or because its function is subserved or complimented by another protein. These studies underscore marked inhibitory pressure and the complexity and plasticity of inhibitory pathways that restrain beta-cell replication.     

Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1

The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.     

Tissue-specific deletion of the retinoblastoma protein in the pancreatic beta-cell has limited effects on beta-cell replication, mass, and function

Animal studies show that G(1/S) regulatory molecules (D-cyclins, cdk-4, p18, p21, p27) are critical for normal regulation of beta-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains beta-cell cycle arrest. Logically, removal of pRb from the beta-cell should result in unrestrained beta-cell replication, increased beta-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in beta-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in beta-cells. Surprisingly, although the pRb gene was efficiently recombined in beta-cells of both CKO models, changes in beta-cell mass, beta-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic beta-cell. This may reflect fundamental inaccuracies in models of beta-cell cycle control or complementation for pRb by undefined proteins.     

Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.     

Increased dye coupling in pancreatic islets from rats in late-term pregnancy

Our previous studies have suggested that elevated lactogen, increased glucose-stimulated insulin secretion, and increased beta-cell coupling are associated. To determine whether this association occurs under conditions of physiologically increased lactogen, we have studied the extent of dye coupling in rat islets during the later stage of pregnancy. These animals have high plasma lactogen levels in the form of placental lactogen, increased plasma insulin, and decreased plasma glucose. The fluorescent tracer, Lucifer yellow CH, was microinjected into central cells of islets from both pregnant and virgin rats, and the extent of transfer was quantitated by determining the projected area of dye spread. Two area measurements were made for each injection, one around the entire discernible fluorescent region ("outer") and another around the distinct brighter region of cells surrounding the injected cell ("inner"). Pregnancy increased dye transfer, as determined by both measurements. The outer area of dye transfer was 9047 +/- 775 microns2 for the islets from pregnant rats and 4699 +/- 391 microns2 for the islets from virgin rats (P less than .001). Similarly, pregnancy increased the inner area of dye transfer, 1447 +/- 161 microns2 for the islets from pregnant rats and 795 +/- 80 microns2 for the islets from virgin rats (P less than .001). These results support the hypothesis that elevated lactogen, increased glucose-stimulated insulin secretion, and increased beta-cell dye coupling are associated under physiological conditions. The study indicates that enhanced beta-cell coupling is part of the structural and functional adaptation that the islets undergo during a subject's pregnancy and demonstrates that the extent of beta-cell coupling is regulated by a physiological condition.     

Characterization of mice doubly transgenic for parathyroid hormone-related protein and murine placental lactogen: a novel role for placental lactogen in pancreatic beta-cell survival

Transgenic overexpression of either parathyroid hormone-related peptide (PTHrP) or mouse placental lactogen type 1 (mPL1) in pancreatic beta-cells, using the rat insulin II promoter (RIP), results in islet hyperplasia either through prolonged beta-cell survival or through increased beta-cell proliferation and hypertrophy, respectively. For determining whether the two proteins might exert complementary, additive, or synergistic effects on islet mass and function when simultaneously overexpressed in beta-cells in vivo, RIP-PTHrP and RIP-mPL1 mice were crossed to generate mice doubly transgenic for PTHrP and mPL1. These double-transgenic mice displayed marked islet hyperplasia (threefold), hypoglycemia, increased beta-cell proliferation (threefold), and resistance to the diabetogenic and cytotoxic effects of streptozotocin compared with their normal siblings. Although the phenotype of the double-transgenic mice was neither additive nor synergistic relative to their single-transgenic counterparts, it was indeed complementary, yielding the maximal salutary phenotypic features of both individual transgenes. Finally, mPL1, for the first time, was shown to exert a protective effect on the survival of beta-cells, placing it among the few proteins that can improve function and proliferation and prolong the survival of beta-cells. Placental lactogen 1 is an attractive target for future therapeutic strategies in diabetes.     

Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass

Overexpression of hepatocyte growth factor (HGF) in the beta-cell of transgenic mice enhances beta-cell proliferation, survival, and function. In the current studies, we have used conditional ablation of the c-met gene to uncover the physiological role of HGF in beta-cell growth and function. Mice in which c-met is inactivated in the beta-cell (MetCKO mice) display normal body weight, blood glucose, and plasma insulin compared with control littermates. In contrast, MetCKO mice displayed significantly diminished glucose tolerance and reduced plasma insulin after a glucose challenge in vivo. This impaired glucose tolerance in MetCKO mice was not caused by insulin resistance because sensitivity to exogenous insulin was similar in both groups. Importantly, in vitro glucose-stimulated insulin secretion in MetCKO islets was decreased by approximately 50% at high glucose concentrations compared with control islets. Furthermore, whereas insulin and glucokinase expression in MetCKO islets were normal, GLUT-2 expression was decreased by approximately 50%. These changes in beta-cell function in MetCKO mice were not accompanied by changes in total beta-cell mass, islet morphology, islet cell composition, and beta-cell proliferation. Interestingly, however, MetCKO mice display an increased number of small islets, mainly single and doublet beta-cells. We conclude that HGF/c-met signaling in the beta-cell is not essential for beta-cell growth, but it is essential for normal glucose-dependent insulin secretion.     

TIMP-1 transgenic mice recover from diabetes induced by multiple low-dose streptozotocin

Type 1 diabetes results from autoimmune destruction of the insulin-producing beta-cells of pancreatic islets, of which the capacity for self-replication in the adult is too limited to restore following extensive tissue injury. Tissue inhibitor of metalloproteinase (TIMP)-1 inhibits matrix metalloproteinase activity and regulates proliferation and apoptosis of a variety of cells types, depending on the context. Here, we show that overexpression of human TIMP-1 in pancreatic beta-cells of transgenic mice counteracts the cytotoxicity and insulitis induced by multiple low-dose streptozotocin (MLDS). Nontransgenic mice developed severe hyperglycemia, hypoinsulinemia, and insulitis 2 weeks after streptozotocin administration and died within 17 weeks. However, MLDS-treated transgenic mice gradually normalized the metabolic parameters and survived. beta-Cell mass increased in parallel as a result of enhancement of beta-cell replication. Thus, our results have demonstrated for the first time that overexpression of TIMP-1 in beta-cells enhances the replication of pancreatic islets beta-cells and counteracts type 1 diabetes, indicating that the TIMP-1 gene may be a potential target to prevent, or even reverse, type 1 diabetes.     

Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways

Cytotoxic T-cells are the major mediators of beta-cell destruction in type 1 diabetes, but the molecular mechanisms are not definitively established. We have examined the contribution of perforin and Fas ligand to beta-cell destruction using islet-specific CD8(+) T-cells from T-cell receptor transgenic NOD8.3 mice. NOD8.3 T-cells killed Fas-deficient islets in vitro and in vivo. Perforin-deficient NOD8.3 T-cells were able to destroy wild-type but not Fas-deficient islets in vitro. These results imply that NOD8.3 T-cells use both pathways and that Fas is required for beta-cell killing only when perforin is missing. Consistent with this theory, transgenic NOD8.3 mice with beta-cells that do not respond to Fas ligation were not protected from diabetes. We next investigated the mechanism of protection provided by overexpression of suppressor of cytokine signaling-1 (SOCS-1) in beta-cells of NOD8.3 mice. SOCS-1 islets remained intact when grafted into NOD8.3 mice and were less efficiently killed in vitro. However, addition of exogenous peptide rendered SOCS-1 islets susceptible to 8.3 T-cell-mediated lysis. Therefore, NOD8.3 T-cells use both perforin and Fas pathways to kill beta-cells and the surprising blockade of NOD8.3 T-cell-mediated beta-cell death by SOCS-1 overexpression may be due in part to reduced target cell recognition.     

Expression of IGF-I in pancreatic islets prevents lymphocytic infiltration and protects mice from type 1 diabetes

Type 1 diabetic patients are diagnosed when beta-cell destruction is almost complete. Reversal of type 1 diabetes will require beta-cell regeneration from islet cell precursors and prevention of recurring autoimmunity. IGF-I expression in beta-cells of streptozotocin (STZ)-treated transgenic mice regenerates the endocrine pancreas by increasing beta-cell replication and neogenesis. Here, we examined whether IGF-I also protects islets from autoimmune destruction. Expression of interferon (IFN)-beta in beta-cells of transgenic mice led to islet beta(2)-microglobulin and Fas hyperexpression and increased lymphocytic infiltration. Pancreatic islets showed high insulitis, and these mice developed overt diabetes when treated with very-low doses of STZ, which did not affect control mice. IGF-I expression in IFN-beta-expressing beta-cells of double-transgenic mice reduced beta(2)-microglobulin, blocked Fas expression, and counteracted islet infiltration. This was parallel to a decrease in beta-cell death by apoptosis in islets of STZ-treated IGF-I+IFN-beta-expressing mice. These mice were normoglycemic, normoinsulinemic, and showed normal glucose tolerance. They also presented similar pancreatic insulin content and beta-cell mass to healthy mice. Thus, local expression of IGF-I prevented islet infiltration and beta-cell death in mice with increased susceptibility to diabetes. These results indicate that pancreatic expression of IGF-I may regenerate and protect beta-cell mass in type 1 diabetes.     

Differential changes of CDK activities in glomeruli and tubules during the active DNA synthetic period after ischemic injury

The present study was designed to determine the spatial correlation among extent of DNA synthetic activity, expressions of G(1)/S phase cyclins, cyclin-dependent kinases (CDKs) and CIP/KIP family of CDK inhibitors (CKIs), and activities of G(1)/S phase CDKs in glomeruli and outer medullae of kidneys during the active regeneration period after ischemic injury. DNA synthetic activity was measured using [(3)H]-thymidine autoradiogram in the kidney sections. Cyclin, CDK, and CKI proteins were determined by Western blot analysis. CDK activities were determined by phosphorylation amount using specific substrate. The protein levels of cyclins (D1, D3, E, A) and activities of CDK4 and CDK2 were increased concomitant with the induction of DNA synthetic activity in outer medullae, but not in glomeruli, in adult kidneys during DNA synthetic period after ischemic injury. The p27(KIP1) protein, but not the p21(CIP1) protein, increased equally in total kidney, glomeruli, and outer medullae after ischemic injury. These results indicate that renal tubules have an active cyclin/CDK system, while glomeruli, do not have a cyclin/CDK system during active regeneration of kidneys after ischemic injury.     

Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced beta-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect beta-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in beta-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced beta-cell death.     

Estrogen can prevent or reverse obesity and diabetes in mice expressing human islet amyloid polypeptide

Type 2 diabetes is characterized by loss of beta-cell mass and concomitant deposition of amyloid derived from islet amyloid polypeptide (IAPP). Previously we have shown that expression of human IAPP (huIAPP) in islets of transgenic mice results in either a rapid onset of hyperglycemia in mice homozygous for the huIAPP transgene on a lean background (FVB/N) or a gradual hyperglycemia in mice hemizygous for the huIAPP transgene on an obese background (A(vy)/A). In both strains, only the males routinely develop diabetes. To investigate this sexual dimorphism, we treated young prediabetic A(vy)/A mice transgenic for huIAPP (huIAPP-A(vy)) with 17beta-estradiol (E2). The treatment completely blocked the progression to hyperglycemia but also prevented the associated weight gain in these mice. Immunohistochemistry of pancreatic sections demonstrated normal islet morphology with no apparent deposition of islet amyloid. E2 treatment of 1-year-old huIAPP-A(vy) diabetic males rapidly reverses obesity and hyperglycemia. To determine the effects of E2 in a nonobese model, we also treated prediabetic, ad libitum-fed and pair-fed Lean-huIAPP transgenic males. E2 completely blocked the progression to hyperglycemia with no significant effect on body weight. Pancreatic insulin content and plasma insulin concentration of Lean-huIAPP transgenic mice increased in a dose-dependent manner. We demonstrated the presence of estrogen receptor (ER)-alpha mRNA in mouse and human islets. By also confirming the presence of ER-alpha protein in islets, we discovered a novel 58-kDa ER-alpha isoform in mice and a 52-kDa isoform in humans, in the absence of the classic 67-kDa protein found in most tissues of both species. The demonstrated presence of ER-alpha in mouse and human islets is consistent with a direct effect on islet function. We conclude that exogenous E2 administered to male mice may block human IAPP-mediated beta-cell loss both by direct action on beta-cells and by decreasing insulin demand through inhibition of weight gain or increasing insulin action.     

Immediate early genes and p21 regulation in liver of rats with acute hepatic failure

It has been observed that liver regeneration in acute hepatic failure (AHF) is suppressed [Eguchi et al. Hepatology 1996;24(6):1452-9]. The molecular mechanism regulating this inhibition is not known. We previously reported that in AHF rats, hepatocyte proliferation was significantly impaired with elevation in serum IL-6, TGF-beta1, and HGF [Kamohara et al. Biochem Biophys Res Commun 2000;273(1):129-35]. Following either 70% partial hepatectomy (PH) or liver injury, quiescent mature hepatocytes are "primed" to re-enter the cell cycle. The process of "priming" appears to be triggered by extracellular cytokines (IL-6 and TNF-alpha) and is characterized by expression of immediate early genes. Under the stimulation of growth factors such as HGF, "primed" hepatocytes exit the G1 phase of the cell cycle. G1-associated cyclins and their inhibitors play a pivotal role in G1/S cell cycle transition. Here, we demonstrate that immediate early gene (i.e. c-myc, c-fos) expression and AP-1 activity are preserved in AHF rat livers despite absence of hepatocyte proliferation. In contrast, p21 mRNA and protein are both over-expressed in AHF livers compared to livers from rats undergoing PH; this elevation leads to inhibition in Cdk2 activity, resulting in G1 cell cycle arrest and inhibition of regeneration.     

Cyclin A和p21cip1在增生性瘢痕成纤维细胞中的表达及与细胞周期相关性的探讨

目的 探讨增生性瘢痕(HS)不同时期成纤维细胞(FB)中细胞周期素A(Cyclin A)、细胞周期蛋白-激酶的抑制因子p21cip1基因及蛋白的表达与同时期FB所处的细胞周期的相关性,为细胞周期相关基因的干预用于HS的防治提供前期理论依据.方法 采集HS不同时段(3、6、12、24个月)瘢痕标本32例,每个时段8例,并以8例正常皮肤作为对照组.利用实时荧光定量PCR法、蛋白质免疫印迹(Western blotting)法检测HS及正常皮肤中Cyclin A和p21cip1的mRNA、蛋白表达水平;用流式细胞术检测HS中FB的细胞周期情况.结果 HS中CyclinA的mRNA及蛋白质表达量在3、6、12、24个月组分别为19.34±2.41、0.99±0.11,19.30±1.42、0.96±0.09,10.73±2.93、0.66±0.58,9.29±0.97、0.65±0.14.p21cip1的mRNA及蛋白质表达量在3、6、12、24个月组分别为2.8±0.69、0.35±0.07;4.95±1.82、0.44±0.07;9.98±1.19、0.56±0.06;10.25±1.46、0.59±0.06.Cyclin A的mRNA和蛋白的表达,3与6个月组比较差异无统计学意义(P>0.05);3、6个月组分别与12、24个月和正常组比较差异有统计学意义(P<0.05);12、24个月与正常组比较差异无统计学意义(P>0.05).p21cip1的mRNA和蛋白的表达,3个月组低于6个月组(P<0.05);3、6个月组分别与12、24个月和正常组比较表达明显降低(P<0.05);12、24个月和正常组比较差异无统计学意义(P>0.05).流式细胞仪检测结果显示3、6个月增生性瘢痕中细胞主要分布在S、G2/M期;而12、24个月组增生性瘢痕及正常皮肤中的细胞多处于G0/G1期.结论 随着HS的发展,HS中CyclinA的mRNA和蛋白的表达强度呈由强至弱的变化,p21cip1的mRNA和蛋白的表达则呈由弱至强的变化.不同时期HS中CyclinA和p21cip1各自的mRNA和蛋白的表达趋势基本一致;不同时期HS中FB的细胞周期分布情况与CyclinA和p21cip1 mRNA和蛋白表达强弱相对应;在HS发生早期对这两个基因进行干预,有可能在控制增生性瘢痕的发生和发展中发挥作用.     

Production and characterization of Reg knockout mice: reduced proliferation of pancreatic beta-cells in Reg knockout mice

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets. We have demonstrated in vitro and in vivo that the exogenous addition of rat and human Reg gene products, Reg/REG proteins, induced beta-cell replication via the Reg receptor and thereby ameliorated experimental diabetes. In the present study, we produced Reg knockout mice by homologous recombination. The Reg gene disruption resulted in a null mutation. Knockout mice developed normally. Islets from the Reg knockout mice appeared morphologically indistinguishable from those of normal controls. However, [(3)H]thymidine incorporation in isolated islets from Reg knockout mice was decreased. When hyperplastic islets were induced by the injection of goldthioglucose, the average islet size in Reg knockout mice was significantly smaller than that of control Reg(+/+) mice. We then produced transgenic mice carrying the Reg gene under the control of the rat insulin II promoter (Ins-Reg) to express Reg in beta-cells. Isolated islets from the Ins-Reg transgenic mice showed increased [(3)H]thymidine incorporation. By intercrossing, we produced NOD mice carrying the Ins-Reg transgene and found that development of diabetes in the resultant Ins-Reg transgenic NOD mice was significantly retarded, coinciding with an increase in the pancreatic beta-cell mass. These results indicate that Reg plays an important role in beta-cell growth/regeneration.     

Spontaneous recovery from hyperglycemia by regeneration of pancreatic beta-cells in Kir6.2G132S transgenic mice

The ATP-sensitive K(+) channel (K(ATP) channel) in pancreatic beta-cells is a critical regulator in insulin secretion. We previously reported that transgenic mice expressing a dominant-negative form (Kir6.2G132S) of Kir6.2, a subunit of the K(ATP) channel, specifically in beta-cells develop severe hyperglycemia in adults (8 weeks of age). In this study, we conducted a long-term investigation of the phenotype of these transgenic mice. Surprisingly, hyperglycemia was spontaneously improved with concomitant improvement of pancreatic insulin content in the transgenic mice at >25 weeks of age. Insulin-positive cells and pancreatic duodenal homeobox 1 (PDX1)-positive cells both were clearly increased in the older compared with the younger transgenic mice. Interestingly, cells labeled with the lectin Dolichos biflorus agglutinin (DBA), a potential indicator of uncommitted pancreatic epithelial/ductal cells, were detected in the islets of the transgenic mice but not in those of wild-type mice. In addition, a subset of the DBA-labeled cells was positive for PDX1, insulin, glucagon, somatostatin, or pancreatic polypeptide. Moreover, some of the DBA-labeled cells were also positive for a proliferating cell marker. These results show that the Kir6.2G132S transgenic mouse is a useful model for studying beta-cell regeneration and that DBA-labeled cells participate in the process.     

外源性一氧化氮对前列腺癌细胞作用的研究

目的研究外源性一氧化氮(NO)对前列腺癌细胞株生长的影响及作用机制.方法以硝普钠(SNP)为一氧化氮供体,不同浓度的SNP作用前列腺癌细胞,采用MTT法测细胞的生存率,Tunel法和流式细胞仪检测细胞的凋亡情况,通过RT-PCR法检测细胞p21^waf1/cip1基因的表达.结果前列腺癌细胞生存率与低浓度的SNP成正比,而与高浓度的SNP成反比.高浓度的SNP可作用于细胞的G1期,诱导细胞凋亡,p21^waf1/cip1的表达也随之上调.结论低浓度的SNP促进前列腺癌细胞的生长;高浓度的SNP促进细胞的凋亡,p21^waf1/cip1的上调可能是高浓度的SNP诱导前列腺癌细胞凋亡的机制之一.